Because of its involvement in a broad range of cellular signal transduction and other mechanisms, the ubiquitin pathway is now recognized as a new opportunity for discovering drugs to treat a variety of diseases. Initial success was achieved with the approval of the proteasome inhibitor Velcade in 2003 for refractory multiple myeloma. It is believed that targeting a less general mechanism than proteasome inhibition would yield drugs with fewer side effects than those seen with Velcade. E3 ligases, the enzymes that add ubiquitins to specific target proteins or groups of proteins, have been studied extensively in the last five years and are considered promising targets for selective drug discovery. Several of them have been linked genetically or biochemically with specific diseases, and assays have been developed to find inhibitors via high throughput screening. Despite this effort, no promising lead compounds have emerged. The goal of this proposal is to develop a simple in vitro E3 ligase assay that is cost effective, detects poly- ubiquitylation, and can be configured for high throughput screening. Poly-ubiquitylation will be detected by using a tagged, quencher-labeled ubiquitin-associated domain (UBA). Such an assay will be superior to those now in use, increasing the likelihood of discovering selective, therapeutically useful inhibitors. The ultimate commercial goal is to develop an assay format that can be applied to various E3 enzymes and substrates, facilitating drug discovery for known and emerging E3-mediated disease processes. Enzymes (called ubiquitin E3 ligases) that add a protein tag known as ubiquitin to key cellular proteins have been linked to various diseases; for this reason, compounds that inhibit relevant E3 ligases are being sought as potential drugs to treat the diseases. This proposal seeks funds to develop a biochemical assay to measure E3 ligase activity; once the assay is developed, it will be adapted to find such inhibitors through high throughput screening. The assay will be based on the destruction of a fluorescence signal when multiple ubiquitins are added to a protein by the E3 ligase and subsequent restoration of the signal when the ligase action is inhibited. In Phase II, compound collections will be screened using this assay, with the aim of finding compounds that have the potential to lead to drugs. [unreadable] [unreadable] [unreadable]